In breast cancer patients, the migration of cancer cells away from the primary tumor and their subsequent metastasis to distant organs is the leading cause of mortality. Metastatic cells escape the primary tumor and enter the bloodstream by developing actin-rich membrane protrusions called invadopodia that degrade the extracellular matrix (ECM) to allow invasion of surrounding tissues. The assembly of invadopodia is regulated by Rho GTPases, a family of proteins that regulates the actin cytoskeleton. However, little is known about how they are activated, the time course of their activation, or the identity of their upstream regulators and downstream effectors. Our long term goal is to characterize the mechanisms of regulation of Rho GTPases that contribute to cancer metastasis, in particular to cancer cell migration and invasion. The objective of this study is to characterize the molecular mechanisms that regulate RhoG-mediated signaling during invadopodia assembly and disassembly. Based on our preliminary results, our central hypothesis is that RhoG functions as a negative regulator of invadopodia formation by regulating the dynamics of their disassembly, in a process that involves the adaptor protein paxillin and yet to be characterized signaling components. We will test our hypothesis by pursuing two specific aims: 1) To characterize the spatio-temporal dynamics of RhoG activation and its role during invadopodia formation and cell invasion. Our hypothesis is that RhoG activation is tightly regulated both temporally and spatially during invadopodia formation and plays a role in their disassembly in a process that requires paxillin phosphorylation. Here, we will use a novel RhoG FRET biosensor to determine the spatiotemporal activation of RhoG during invadopodia formation in live cells. We will also characterize the role of RhoG in the regulation of invadopodia dynamics, degradation of ECM and cell invasion. 2) To identify the RhoG-specific effectors involved in invadopodia formation. The objective for this Aim is to characterize the immediate RhoG downstream effectors and their role during invadopodia formation. We have identified several potential RhoG-effectors using a novel proximity-based labeling assay coupled to mass spectrometry. Based on our preliminary results, our working hypothesis is that some of these RhoG-specific effectors are critical for the regulation of invadopodia formation. The work proposed here is expected to shed light on a novel signaling pathway that plays a role in the regulation of invadopodia assembly and function. Such results are expected to have an important positive impact, because they may identify novel targets that could drive the development of therapeutics to prevent or slow the progression of breast cancer metastasis, in addition to fundamentally advance the fields of cancer cell migration and invasion.